ZEISS
Level-adjustable Sample Holder with Insert for Piezo Stage
ELYRA 7
18
000000-2262-999
03/2019 V_02
d)
Use the front right screw (Fig. 6/
sy
) of the sample holder to readjust the focus (Fig. 7/
E
). Turn
clockwise if the focus position was positive, counter clockwise if it was negative. (Note that one
turn corresponds to approximately 125 µm).
e)
Press the lower pointing arrow of the
Y
input box of the
Stage
tool to return to the initial
position. If the image is in focus (Fig. 7/
F
) Y leveling is complete; otherwise repeat steps 4a) - e)
iteratively. If applicable, the multiplication factor of 4c) can be adjusted.
5.3
Stage Leveling Using X-Cite in Reflection
Fig. 11
Adjustable sample holder –
schematic view
Fig. 12
Focus control in reflection
1.
Assemble a fluorescent sample you want to level with sample bottom downwards into the sample
holder. The sample site should be clean and free of any immersion oil. Position the sample such that
the objective lens is in the middle of the sample. Use WF illumination with the X-Cite at 12 % and
filter for excitation with the 561 nm line (optimal would be a small band pass to avoid too much
light). Swing nosepiece to empty position (make sure no cover is in the empty position) and use
preferentially Andor SIM camera, albeit the Andor PALM camera would also work. Take Optovar 1.0
and set EM gain to zero.
2.
Draw fiber plug of X-Cite completely out from the X-Cite casing. Start acquisition. The image will be
dark since fiber was pulled out. Put fiber back and slowly move in until you will see a coarsely
adjusted image to have sufficiently reduced light levels you can work with (Fig. 12/
A
). (Note: with
the fiber all in, the lamp is simply to bright leading to oversaturation of the camera). If the
illumination is far from being centric, the sample is already strongly tilted and it is recommended to
do a pre-adjustment. To this end adjust the edges of the clefts viewed from the right side
(Fig. 11/
csv
) and back side (Fig. 11/
cbv
) to be parallel using screws
sy
and
sx
, respectively (Fig. 11).
The cleft’s width should be approximately 2 mm as preset by the factory, but can be adjusted with
screw
sz
if needed.
3.
Now pull (or push) fiber on the ELYRA site until the dark ring, which is already faintly visible in its
contours, appears focused (Fig. 12/
B
). Dependent on the sample the focus could look worse,
especially when the reflecting planes are wavy or you get reflection from different planes (Fig. 12/
C
).
4.
Move in 10x objective and focus the already visible ring with the z-drive of the stand. The ring
appears now well focused as long as the sample is not completely unsuitable (Fig. 12/
D
)